Thermo Fisher Scientific TaqMan Copy Number Assays User guide
- Type
- User guide
For Research Use Only. Not for use in diagnostic procedures.
TaqMan™ Copy Number Assays
USER GUIDE
Publication Number 4397425
Revision F
Manufacturer:
Life Technologies Holdings Pte Ltd |
Block 33 |
Marsiling Industrial Estate Road 3 |
#07-06, Singapore 739256
Products:
TaqMan Copy Number Assays
Custom TaqMan Copy Number Assays
Custom Plus TaqMan Copy Number Assays
TaqMan Copy Number Reference Assay, human, RNase P
TaqMan Copy Number Reference Assay, human, TERT
TaqMan Copy Number Reference Assay, mouse, Tfrc
TaqMan Copy Number Reference Assay, mouse, TERT
Manufacturer:
Life Technologies Corporation |
6055 Sunol Blvd |
Pleasanton, CA 94566
Products:
TaqMan Copy Number Assays
Custom TaqMan Copy Number Assays
Custom Plus TaqMan Copy Number Assays
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.
Revision history: Pub. No. 4397425
Revision Date Description
F 10 December 2019 Removed troubleshooting and replaced with link to FAQs.
E 6 February 2019 • Added new instruments, Master Mixes, and other applicable
products.
• Added details for dilution of 60X assays.
• Added storage information for gDNA.
• Updated chemistry information.
• Updated order and design information.
• Updated for general style, formatting, and branding.
D September 2010 Baseline for this revision history.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered
trademark of Roche Molecular Systems, Inc., used under permission and license. AmpErase is a trademark of Roche Molecular Systems, Inc.
©2019 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 Product information ....................................... 5
Product description ............................................................. 5
Assay description ........................................................... 6
Contents and storage ............................................................ 8
TaqMan™ Copy Number Assays ............................................... 8
Reference assay description ................................................. 8
Order copy number assays and reference assays .................................... 9
Master Mixes ................................................................... 9
Required materials not supplied ................................................. 10
Workflow ..................................................................... 12
■CHAPTER 2 Guidelines for preparation of gDNA ..................... 13
Guidelines for isolation of high–quality gDNA ...................................... 13
Guidelines to quantify the gDNA ................................................. 13
■CHAPTER 3 Perform PCR amplification .............................. 14
Determine the number of sample types and replicates .............................. 14
Dilute the 60X assays ........................................................... 14
Prepare the PCR reactions ...................................................... 14
Prepare the PCR reactions with wet gDNA .................................... 15
Prepare the PCR reactions with dried–down gDNA ............................. 16
Set up and run the real–time PCR instrument ...................................... 17
■CHAPTER 4 Analyze and export results .............................. 19
Analyze the results ............................................................. 19
Export the results .............................................................. 19
Guidelines to export the results ............................................. 20
Import the results to the CopyCaller™ Software ................................... 20
TaqMan
™
Copy Number Assays User Guide
3
■APPENDIX A Troubleshooting and FAQs .............................. 21
■APPENDIX B Supplemental information .............................. 22
TaqMan™ chemistry ............................................................ 22
About the 5’ nuclease assay ................................................. 22
Best practices for PCR and RT-PCR experiments ................................... 24
Good laboratory practices for PCR and RT-PCR ................................ 24
Use UNG to prevent false-positive amplification ............................... 24
Detect fluorescent contaminants ............................................ 25
■APPENDIX C Safety ..................................................... 26
Chemical safety ................................................................ 27
Biological hazard safety ......................................................... 28
■Documentation and support ............................................. 29
Related documentation ......................................................... 29
Customer and technical support ................................................. 30
Limited product warranty ....................................................... 30
Contents
4
TaqMan
™
Copy Number Assays User Guide
Product information
■Product description .................................................... 5
■Contents and storage .................................................. 8
■Order copy number assays and reference assays ........................... 9
■Master Mixes ......................................................... 9
■Required materials not supplied ........................................ 10
■Workow ........................................................... 12
Product description
Applied Biosystems™ TaqMan™ Copy Number Assays are designed to detect and
measure copy number variation within the human and mouse genomes. Copy
number variation is an important polymorphism associated with genetic diseases
such as cancer, immune diseases, and neurological disorders.
• TaqMan™ Copy Number Assays
– Include an extensive collection of pre–designed assays.
– Are available for genes and known copy number variation regions.
– Are available for human and mouse genomes.
– Provide common vector marker and reporter genes for study of transfected
cells and organisms.
• Custom Plus TaqMan™ Copy Number Assays
– Include bioinformatics analysis on the target and assay sequences.
– Can be designed for other targets of interest in human and mouse genomes.
• Custom TaqMan™ Copy Number Assays
– Can be designed for other targets of interest.
– Do not include bioinformatic analysis.
• TaqMan™ Copy Number Reference Assays
– Are designed to unique human and mouse genomic sequences.
– Are required for relative quantitation of copy number targets.
This document provides instructions for performing and analyzing copy number
variation quantication experiments using the copy number assays and the copy
number reference assays.
1
TaqMan
™
Copy Number Assays User Guide
5
The copy number assays are run simultaneously with a copy number reference assay
in a duplex real-time polymerase chain reaction (PCR). The copy number assay
detects the target gene or genomic sequence of interest, and the copy number
reference assay detects a sequence that is known to exist in two copies in a diploid
genome, for example, the human RNase P H1 RNA gene, RPPH1. This method of
relative quantitation is used to determine the relative copy number of the target of
interest in a genomic DNA (gDNA) sample, normalized to the known copy number of
the reference sequence.
The number of copies of the target sequence in each test sample is determined by
relative quantitation (RQ) using the comparative Ct (ΔΔCt) method. This method
measures the Ct dierence (ΔCt) between target and reference sequences, then
compares the ΔCt values of test samples to a calibrator sample known to have two
copies of the target sequence. The copy number of the target is calculated to be two
times the relative quantity.
In a copy number quantication reaction, puried gDNA is combined with the copy
number assay and the copy number reference assay.
The copy number assay includes the following components:
• Two unlabeled primers for amplifying the target sequence of interest.
• One TaqMan™ MGB probe for detecting the target sequence of interest. The probe
incorporates the following items:
– A FAM™ reporter dye, aached to the 5’ end.
– A nonuorescent quencher (NFQ) and a Minor Groove Binder (MGB),
aached to the 3’ end.
Note: MGBs increase the melting temperature (Tm) without increasing probe
length. They allow for the design of shorter probes.
The copy number reference assay includes the following components:
• Two unlabeled primers for amplifying the reference sequence.
• One TaqMan™ TAMRA™ probe for detecting the reference sequence. The probe
incorporates the following items:
– A VIC™ reporter dye, aached to the 5’ end.
– A TAMRA™ quencher, aached to the 3’ end.
Table 1 Description of the reference assays
Assay Purpose Function
Human
TaqMan™ Copy Number Reference
Assay, human, RNase P
Standard reference assay for human
gDNA copy number quantitation
experiments
Detects the Ribonuclease P RNA
component H1 (H1RNA) gene
(RPPH1) on chromosome 14,
cytoband 14q11.2.
TaqMan™ Copy Number Reference
Assay, human, TERT
Alternative reference assay for
human gDNAs
Targets the telomerase reverse
transcriptase (TERT) gene located on
chromosome 5, cytoband 5p15.33.
Assay description
Chapter 1 Product information
Product description
1
6
TaqMan
™
Copy Number Assays User Guide
Assay Purpose Function
Mouse
TaqMan™ Copy Number Reference
Assay, mouse, Tfrc
Standard reference assay for mouse
gDNA copy number quantitation
experiments
Detects the transferrin receptor gene
(Tfrc) on chromosome 16, cytoband
16qB3.
TaqMan™ Copy Number Reference
Assay, mouse, TERT
Alternative reference assay for mouse
gDNA
Targets the telomerase reverse
transcriptase (TERT) gene located on
chromosome 13, cytoband 13qC1.
Chapter 1 Product information
Product description
1
TaqMan
™
Copy Number Assays User Guide
7
Contents and storage
Each assay contains target-specic forward and reverse primers and FAM™ dye-
labeled MGB probe.
All of the assays are available in 20✕ and 60✕ concentrations and are made to order.
Table 2 TaqMan™ Copy Number Assays
Size
Number of reactions Cat. No.
Shipping and
storage
10–µL reaction 20–µL reaction
TaqMan™
Copy
Number
Assays
Custom
TaqMan™
Copy
Number
Assays
Custom Plus
TaqMan™ Copy
Number Assays
Small (20X) 720 360 4400291 4400294 4442487 Shipped at
ambient
temperature.
Store at −25°C
to −15°C.
Minimize
freeze–thaw
cycles.
Medium
(20X) 1500 750 4400292 4400295 4442520
Large (60X) 5800 2900 4400293 4400296 4442488
Each reference assay detects a single-copy gene in its respective reference genome
assembly. Each reference assay contains reference sequence-specic forward and
reverse primers and a VIC™ dye-labeled TAMRA™ probe.
The reference assays are ordered separately from the copy number assays. For a
description of the reference assays, see Table 1 on page 6.
Table 3 Reference assays
Product
Number of reactions
Cat. No. Shipping and storage
10–µL reactions 20–µL
reactions
Human
TaqMan™ Copy Number
Reference Assay, human, RNase
P, 1 tube (20X)
1500 750 4403326
Shipped at ambient
temperature.
Store at −25°C to −15°C.
Minimize freeze–thaw
cycles.
TaqMan™ Copy Number
Reference Assay, human, RNase
P, 4 tubes (20X)
6000 3000 4403328
TaqMan™ Copy Number
Reference Assay, human, TERT, 1
tube (20X)
1500 750 4403316
TaqMan™ Copy Number
Reference Assay, human, TERT, 4
tubes (20X)
6000 3000 4403315
TaqMan™ Copy
Number Assays
Reference assay
description
Chapter 1 Product information
Contents and storage
1
8
TaqMan
™
Copy Number Assays User Guide
Product
Number of reactions
Cat. No. Shipping and storage
10–µL reactions 20–µL
reactions
Mouse
TaqMan™ Copy Number
Reference Assay, mouse, Tfrc, 1
tube (20X)
1500 750 4458366
Shipped at ambient
temperature.
Store at −25°C to −15°C.
Minimize freeze–thaw
cycles.
TaqMan™ Copy Number
Reference Assay, mouse, Tfrc, 4
tubes (20X)
6000 3000 4458367
TaqMan™ Copy Number
Reference Assay, mouse, TERT, 1
tube (20X)
1500 750 4458368
TaqMan™ Copy Number
Reference Assay, mouse, TERT, 4
tubes (20X)
6000 3000 4458369
Order copy number assays and reference assays
• Order TaqMan™ Copy Number Assays.
a. Go to thermosher.com/taqmancnv.
b. Use the Assay Search Tool.
• Design and order Custom TaqMan™ Copy Number Assays and Custom Plus
TaqMan™ Copy Number Assays, and order TaqMan™ Copy Number Reference
Assays.
a. Go to thermosher.com/taqmancustomcnv.
b. Read the background material, then click Order Custom TaqMan™ Copy
Number Assays.
c. Follow the prompts to search for or enter the sequences you need.
Master Mixes
The following Master Mixes are recommended for copy number assays:
• TaqPath™ ProAmp™ Master Mix for samples with PCR inhibitors such as heparin
and hematin
• TaqMan™ Genotyping Master Mix
Chapter 1 Product information
Order copy number assays and reference assays
1
TaqMan
™
Copy Number Assays User Guide
9
Table 4 TaqPath™ ProAmp™ Master Mix
Cat. No. Amount Storage
A30865 1 x 1 mL
2–8°C
A30866 1 x 10 mL
A30871 2 x 10 mL
A30867 1 x 50 mL
A30872 2 x 50 mL
Table 5 TaqMan™ Genotyping Master Mix
Cat. No. Amount Storage
4371353 1 x 1 mL
2–8°C
4371355 1 x 10 mL
4381656 2 x 10 mL
4371357 1 x 50 mL
4381657 2 x 50 mL
The following Master Mixes are also compatible with copy number assays:
• TaqMan™ Gene Expression Master Mix
• TaqMan™ Universal PCR Master Mix
• TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG
• TaqMan™ Universal Master Mix II
For details on PCR conditions for the Master Mixes see the appropriate user guide.
Note: TaqMan™ Fast Advanced Master Mix is not recommended for use with copy
number assays.
Required materials not supplied
Unless otherwise indicated, all materials are available through thermosher.com.
MLS: Fisher Scientic (sherscientic.com) or other major laboratory supplier.
Table 6 Recommended products to prepare gDNA
Item Source
Kits to prepare gDNA thermofisher.com/
gdnaprep
Chapter 1 Product information
Required materials not supplied
1
10
TaqMan
™
Copy Number Assays User Guide
Table 7 Other materials and equipment required for the workflow
Item Source
Real-time PCR instrument, one of the following:
QuantStudio™ 3 or 5 Real-Time PCR System
Contact your local
sales office
QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System
QuantStudio™ 12K Flex Real–Time PCR System
StepOnePlus™ Real-Time PCR System
ViiA™ 7 Real-Time PCR System
7500/7500 Fast Real-Time PCR System
7900HT Fast Real‑Time PCR System
Software
CopyCaller™ Software (recommended to analyze copy number
assays)
https://
www.thermofisher.co
m/us/en/home/
technical-resources/
software-downloads/
copycaller-
software.html
Equipment
Centrifuge, with adapter for 96-well plates or 384-well plates MLS
Microcentrifuge MLS
Vortex mixer MLS
Pipettes MLS
Tubes, plates, and other consumables
Tubes, plates, and film thermofisher.com/
plastics
Aerosol-resistant barrier pipette tips MLS
Disposable gloves MLS
Reagents
Nuclease-free Water AM9930
TaqMan™ DNA Template Reagents 401970
TaqMan™ RNase P Detection Reagents Kit 4316831
(Optional)
TE, pH 8.0 AM9849
Chapter 1 Product information
Required materials not supplied
1
TaqMan
™
Copy Number Assays User Guide
11
Workflow
Start with gDNA (see page 13)
▼
Determine the number of sample types and replicates (page 14)
▼
Prepare the PCR reactions (page 14)
▼
Set up and run the real–time PCR instrument (page 17)
▼
Analyze and export results (page 19)
Chapter 1 Product information
Workflow
1
12
TaqMan
™
Copy Number Assays User Guide
Guidelines for preparation of gDNA
Guidelines for isolation of high–quality gDNA
The target template for copy number assays is puried genomic DNA (gDNA). Wet
gDNA or dried-down gDNA can be used. For recommended DNA isolation kits see
Table 6 on page 10.
Guidelines to quantify the gDNA
Quantication of gDNA is recommended. Use one of the following methods:
• The method described in RNase P Quantication for Genotyping Experiments (Pub.
No. MAN0014349 ).
• UV absorbance (A260/A280) measurements. An A260/A280 ratio of greater than 1.7
is recommended for human and mouse gDNA.
The RNase P method is preferred because it is more accurate than UV absorbance,
and it assesses sample quality.
2
TaqMan
™
Copy Number Assays User Guide
13
Perform PCR amplification
■Determine the number of sample types and replicates ..................... 14
■Dilute the 60X assays ................................................. 14
■Prepare the PCR reactions ............................................. 14
■Set up and run the real–time PCR instrument ............................ 17
Determine the number of sample types and replicates
The following samples are recommended on each plate:
•Samples or Unknowns—gDNA samples in which the copy number of the target
is unknown.
•No–Template Controls (NTC)—A sample that does not contain a DNA template.
It shows the background uorescence and allows for the detection of
contamination.
•Reference sample—A DNA sample with a known copy number for the target of
interest.
Four replicates of each gDNA sample are recommended for reliable copy number
calls.
Dilute the 60X assays
Dilute 60X assays to 20X with TE, pH 8.0. Store the diluted samples at 2–8°C for
immediate use or at −25°C to −15°C for long-term storage. Minimize exposure to light.
Prepare the PCR reactions
Copy number assays can be used with either wet or dried-down DNA. Drying down
the gDNA in plates is the most convenient method for an experiment that requires
multiple plates using the same gDNA or for multiple experiments that use the same
gDNA.
Select the workow to prepare PCR reactions based on whether wet gDNA or dried–
down gDNA is used.
3
14
TaqMan
™
Copy Number Assays User Guide
Normalize the wet gDNA sample concentration
Dilute each sample to 5 ng/µL with nuclease–free water or 1✕ TE buer, pH 8.
This is a 5✕ stock solution.
Store the diluted samples at 4°C for immediate use or at −25°C to −15°C for long-term
storage.
Prepare the PCR Reaction Mix
Thaw the copy number assays and the copy number reference assays. Gently vortex
to mix, then centrifuge briey to bring contents to the boom of the tube.
1. Mix the Master Mix thoroughly but gently.
2. In an appropriately-sized centrifuge tube, prepare PCR Reaction Mix according
to the following table.
Component
Volume per well[1]
384–well (0.1–mL) or
96–well fast (0.1–mL)
plate
96–well standard (0.2–
mL) plate
Master Mix (2X) 5.5 µL 11.0 µL
TaqMan™ Copy Number Assay
(20✕)0.55 µL 1.1 µL
TaqMan™ Copy Number
Reference Assay (20✕)0.55 µL 1.1 µL
Nuclease-free Water 2.2 µL 4.4 µL
Total PCR Reaction Mix volume 8.8 µL 17.6 µL
[1] Includes 10% overage.
3. Vortex the tube to mix the PCR Reaction Mix thoroughly, then centrifuge briey
to collect the contents at the boom of the tube.
4. Add the PCR Reaction Mix to the appropriate wells of the plate.
• 384–well (0.1–mL) or 96–well fast (0.1–mL) plate—8 µL per well
• 96–well standard (0.2–mL) plate—16 µL per well
5. Gently vortex the gDNA samples, then add the gDNA to the appropriate wells of
the plate.
• 384–well (0.1–mL) or 96–well fast (0.1–mL) plate—2 µL of gDNA per well
• 96–well standard (0.2–mL) plate—4 µL of gDNA per well
6. Seal the reaction plate with optical adhesive lm, then vortex briey to mix the
contents.
7. Centrifuge the plate briey to collect the contents at the boom of the well.
Proceed to “Set up and run the real–time PCR instrument“ on page 17.
Prepare the PCR
reactions with wet
gDNA
Chapter 3 Perform PCR amplification
Prepare the PCR reactions
3
TaqMan
™
Copy Number Assays User Guide
15
Dilute and dry the gDNA
The same amout of gDNA must be added to the reaction plate to dry. However,
gDNA samples do not need to be diluted to the same concentration to dry.
1. Transfer the gDNA into each well of a plate.
• 384–well (0.1–mL) plate or 96–well fast (0.1–mL) plate—10 ng per well
• 96–well standard (0.2–mL) plate—20 ng per well
2. Allow the sample to dry at room temperature in an amplicon-free location.
Store the diluted samples at 4°C for immediate use or at −25°C to −15°C for long-term
storage.
Prepare the PCR reactions
Thaw the copy number assays and the copy number reference assays. Gently vortex
to mix, then centrifuge briey to bring contents to the boom of the tube.
1. Mix the Master Mix thoroughly but gently.
2. In an appropriately-sized centrifuge tube, prepare PCR Reaction Mix according
to the following table.
Component
Volume per well[1]
384–well (0.1–mL) or
96–well fast (0.1–mL)
plate
96–well standard (0.2–
mL) plate
Master Mix (2X) 5.5 µL 11.0 µL
TaqMan™ Copy Number Assay
(20✕)0.55 µL 1.1 µL
TaqMan™ Copy Number
Reference Assay (20✕)0.55 µL 1.1 µL
Nuclease-free Water 4.4 µL 8.8 µL
Total PCR Reaction Mix volume 11.0 µL 22.0 µL
[1] Includes 10% overage.
3. Vortex the tube to mix the PCR Reaction Mix thoroughly, then centrifuge briey
to collect the contents at the boom of the tube.
4. Add the PCR Reaction Mix to each well of the reaction plate containing the
dried–down gDNA.
• 384–well (0.1–mL) or 96–well fast (0.1–mL) plate—10 µL per well
• 96–well standard (0.2–mL) plate—20 µL per well
5. Seal the reaction plate with optical adhesive lm, then vortex briey to mix the
contents.
6. Centrifuge the plate briey to collect the contents at the boom of the well.
Proceed to “Set up and run the real–time PCR instrument“ on page 17.
Prepare the PCR
reactions with
dried–down gDNA
Chapter 3 Perform PCR amplification
Prepare the PCR reactions
3
16
TaqMan
™
Copy Number Assays User Guide
Note: If the plate is not run immediately, protect it from light and store at 2–8°C. If
necessary, the plate can be stored for up to 72 hours if using TaqPath™ ProAmp Master
Mix.
Set up and run the real–time PCR instrument
See the appropriate instrument user guide for detailed instructions to program the
thermal cycling conditions or to run the plate.
Note: The instrument must be congured with the block appropriate for the plate
type.
1. Set the experiment properties.
• Block—384–well or 96–well fast (0.1 mL) or 96–well standard (0.2 mL)
• Experiment type—Standard curve
• Reagents—TaqMan Reagents
• Properties—Standard
2. Dene the targets and the samples, detectors, reporters and quenchers.
Table 8 Required instrument setup information
Instrument Detector
name Target name Reporter Quencher
For copy number assays
7500/7500 Fast Real-Time PCR Systems (software v2.
X
)
QuantStudio™ 3 and 5 Real-Time PCR Systems
QuantStudio™ 6 and 7 Flex Real-Time PCR Systems
QuantStudio™ 12K Flex Real–Time PCR System
ViiA™ 7 Real-Time PCR System
StepOnePlus™ Real-Time PCR System
NA User–
defined FAM™ dye MGB–NFQ
7500/7500 Fast Real-Time PCR Systems (software
v1.
X
)[1] FAM™ dye N/A FAM™ dye None
7900HT Fast Real–Time PCR Systems (software v2.
X
)User–
defined N/A FAM™ dye Nonfluorescent
For copy number reference assays
7500/7500 Fast Real-Time PCR Systems (software v2.
X
)
QuantStudio™ 3 and 5 Real-Time PCR Systems
QuantStudio™ 6 and 7 Flex Real-Time PCR Systems
QuantStudio™ 12K Flex Real–Time PCR System
ViiA™ 7 Real-Time PCR System
StepOnePlus™ Real-Time PCR System
NA User–
defined VIC™ dye TAMRA™ probe
7500/7500 Fast Real-Time PCR Systems (software
v1.
X
)[1] VIC™ dye NA VIC™ dye TAMRA™ probe
7900HT Fast Real–Time PCR Systems (software v2.
X
)User–
defined NA VIC™ dye TAMRA™ probe
[1] For SDS Software v1.
X
, specify FAM™ and VIC™ as the detector names for the copy number assay. SDS Software v1.
X
does not export dye
information, so the reporter dyes must be specified in the detector name.
Chapter 3 Perform PCR amplification
Set up and run the real–time PCR instrument
3
TaqMan
™
Copy Number Assays User Guide
17
3. Assign the targets and samples to each well.
4. Set up the thermal protocol.
Note: PCR conditions are the same for TaqPath™ ProAmp™ Master Mix and
TaqMan™ Genotyping Master Mix. TaqPath™ ProAmp™ Master Mix contains
heat–labile UNG, which is active during the reaction setup and is completely
inactivated during the rst ramp to the 95°C hold step.
Step Temperature Time Cycles
Initial denature / enzyme
activation 95°C 10 minutes 1
Denature 95°C 15 seconds 40
Anneal / Extend 60°C 60 seconds
5. Load the plate into the real–time PCR instrument.
6. Start the run.
Chapter 3 Perform PCR amplification
Set up and run the real–time PCR instrument
3
18
TaqMan
™
Copy Number Assays User Guide
Analyze and export results
■Analyze the results ................................................... 19
■Export the results .................................................... 19
■Import the results to the CopyCaller™ Software .......................... 20
Analyze the results
On the instrument, open the Analysis Seings window and use the following
seings:
• Manual Ct threshold: 0.2
• Autobaseline: On
Apply the seings, close the window, and run the experiment analysis.
For detailed information about data analysis, see the appropriate documentation for
your instrument.
The general guidelines for copy–number data analysis include the following
parameters:
• Ensure that the amplication curves for the copy number reference assay (VIC™
dye signal) in all samples have a distinct linear amplication phase.
• Ensure that the amplication curves for the copy number assay (FAM™ dye
signal) in most wells have a distinct linear amplication phase.
Note: Samples that contain zero copies of the target of interest do not amplify
well, if at all, with the copy number assay. Such samples have high or
undetermined FAM™ Ct values.
• Review any displayed quality check (QC) ags, then review the real-time data of
the associated samples.
Export the results
Export the results les for analysis with the CopyCaller™ Software. For detailed
instructions, see CopyCaller™ Software v2.0 User Guide (Pub. No. 4400042).
Note: If using the ViiA™ 7 Software v1.0 and the CopyCaller™ Software v2.0 for copy
number data analysis, export results using the 7900 Format option. The CopyCaller™
Software cannot open ViiA™ 7 Software v1.0 export les in other formats.
IMPORTANT! Do not modify the exported data les. The CopyCaller™ Software
might not be able to import les that have been modied.
4
TaqMan
™
Copy Number Assays User Guide
19
IMPORTANT! If you run multiple copy number assays or copy number reference
assays on each plate, apply distinct assay detector and target names to the document.
See details in “Set up and run the real–time PCR instrument“ on page 17. User-
dened target and detector names allow the CopyCaller™ Software to arrange and
analyze data independently for each assay in a single exported le.
IMPORTANT! If you use a 7500/7500 Fast Real-Time PCR System running SDS
Software v1.X, you must specify FAM™ dye and VIC™ dye as the detector names for
the copy number assays and copy number reference assays, respectively. SDS
Software v1.X does not export dye information, so you must specify the reporter dye
in the detector name.
Assays per
plate
Assay placement
specified with
separate target
and detector
Procedure
1 — Export the real-time PCR results to a tab-delimited
text (TXT) file or comma-separated values (CSV) file.
>1
Yes
Export the real-time PCR results to a single
exported file (CSV or TXT) that includes all wells of
the plate. The CopyCaller™ Software uses the target
and detector names to distinguish the data from the
different assays.
No
Export the real-time PCR results of each assay to a
separate exported file.
1. Select the wells of the plate that contain the
data from one of the copy number assays.
2. Select File4Export, then export the data from
the selected wells to a data file.
3. Repeat to export the data from the other
assays present on the plate.
Note: To help with organization, name each
exported file according to the assay data it contains.
Import the results to the CopyCaller™ Software
For more information, see the CopyCaller™ Software v2.0 User Guide (Pub. No. 4400042).
1. Import the exported real-time PCR results le into the software.
2. Run the analysis to determine the copy number for your target in each sample.
Guidelines to
export the results
Chapter 4 Analyze and export results
Import the results to the CopyCaller
™
Software
4
20
TaqMan
™
Copy Number Assays User Guide
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
-
1
-
2
-
3
-
4
-
5
-
6
-
7
-
8
-
9
-
10
-
11
-
12
-
13
-
14
-
15
-
16
-
17
-
18
-
19
-
20
-
21
-
22
-
23
-
24
-
25
-
26
-
27
-
28
-
29
-
30
-
31
-
32
Thermo Fisher Scientific TaqMan Copy Number Assays User guide
- Type
- User guide
Ask a question and I''ll find the answer in the document
Finding information in a document is now easier with AI
Related papers
-
Thermo Fisher Scientific QuantStudio 6 and 7 Flex Real-Time PCR System User guide
-
Thermo Fisher Scientific Plus/Minus Assay: Applied Biosystems 7900HT Fast Real-Time PCR Systems Owner's manual
-
Thermo Fisher Scientific TaqPath 1-Step Multiplex Master Mix User guide
-
Thermo Fisher Scientific TaqMan Pri‑miRNA Assays User guide
-
Thermo Fisher Scientific TaqMan® Universal PCR Master Mix User guide
-
Thermo Fisher Scientific TaqMan Gene Expression Assays User guide
-
Thermo Fisher Scientific QuantStudio 3D AnalysisSuite Software User guide
-
Thermo Fisher Scientific TaqMan Fast Advanced Master Mix User guide
-
Thermo Fisher Scientific TaqPath ProAmp Master Mixes User guide
-
Thermo Fisher Scientific 7300/7500/7500 FAST REAL TIME PCR SYSTEM User guide
